ARTIGO ARTICLE 463

Occurrence of imposex in haemastoma: possible evidence of environmental contamination derived from organotin compounds in Rio de Janeiro and Fortaleza, Brazil

Ocorrência de imposex em Thais haemastoma: Marcos Antonio Fernandez 1,2 possíveis evidências de contaminação ambiental Aricelso Maia Limaverde 1 por compostos organotínicos no Rio de Janeiro Italo Braga de Castro 3 e em Fortaleza, Brasil Ana Cristina Martins Almeida 1 Angela de Luca Rebello Wagener 1

1 Departamento de Química, Abstract There are indications that the widespread use of organotin compounds (TBT and TPT) Pontifícia Universidade as antifoulings, as stabilizers in plastic and as pesticides, has severely affected several of Católica do Rio de Janeiro. Rua Marquês de São Vicente marine organisms. The most striking effect of TBT and TPT as hormonal disruptors is the devel- 225, Rio de Janeiro, RJ opment of male organs in females of gastropods, currently denominated imposex. This syndrome 22453-900, Brasil. can lead to the sterilization and death of affected organisms. The present work gives an overview 2 Departamento de Oceanografia, Universidade of the present state of knowledge on imposex occurrence and reports results of a survey conduct- do Estado do Rio de Janeiro. ed in Guanabara , Rio de Janeiro and in several sites along the coast of Fortaleza, Ceará State. Rua São Francisco Xavier Different stages of imposex development were verified in this survey, however, the most promi- 524, sala 4018E, Rio de Janeiro, RJ nent levels appeared associated to known spot sources of TBT and TPT. 20550-013, Brasil. Key words Organotin Compounds; Thais haemastoma; Imposex; Endocrine Disruptors [email protected] 3 Departamento de Biologia, Laboratório de Malacologia, Resumo Existem evidências de que a utilização amplamente disseminada de compostos orga- Universidade Federal noestânicos (TBT e TPT) como antiincrustantes, estabilizadores em plásticos e como pesticidas do Ceará. Av. Humberto Monte s/n, Campus do Pici, tenha afetado severamente diversas espécies de organismos marinhos. O mais característico Bloco 909, Fortaleza, CE efeito do TBT e do TPT como desreguladores endócrinos é o desenvolvimento de caracteres sexuais 60000-000, Brasil. masculinos em fêmeas de gastrópodos, conhecido como imposex. Esta síndrome pode levar à es- terelização e morte dos organismos afetados. O presente trabalho apresenta uma vista geral ao estado atual do conhecimento sobre a ocorrência do imposex, e reporta os resultados de um es- tudo conduzido na Baía de Guanabara, Rio de Janeiro, e ao longo da costa de Fortaleza, no Ceará. Diferentes estágios de desenvolvimento do imposex foram verificados neste estudo, os mais proeminentes parecendo associados às fontes pontuais locais conhecidas de TBT e TPT. Palavras-chave Compostos Orgânicos de Estanho; Thais haemastoma; Imposex; Desregulado- res Endócrinos

Cad. Saúde Pública, Rio de Janeiro, 18(2):463-476, mar-abr, 2002 464 FERNANDEZ, M. A. et al.

Introduction the International Maritime Organization (IMO) recommended the global banning of organotin Organotin compounds have been in use since compounds as antifoulings by 2008 and the the 1960s. Due to its powerful antifouling effect prohibition of new application in boats by 2003 tributyltin (TBT) has been utilized as an addi- (Champ, 1999; ten Hallers-Tjabbes, 1997). In tive in paintings applied to ships, fishing nets, Brazil, however, there is no legislation impos- marine installations and cooling systems based ing restriction or addressing permissible levels, on seawater (Bacci & Gaggi, 1989; Fent, 1996; and up to very recently no investigations have Horiguchi et al., 1994; Short & Thrower, 1986). been conducted to evaluate possible environ- Triphenyltin (TPT) has been used in associa- mental impacts. tion with TBT in antifouling paintings but is al- By the beginning of the last decade, restric- so effectively applied as acaricide and fungi- tions imposed to the use of TBT and TPT in de- cide in agriculture and for wood preservation veloped countries had led to the decrease in (Favoreto, 2000; Fent, 1996; Mensink et al., concentration of the parent compounds and 1997). Di-substituted organotin compounds are decay products, both in waters and organisms. used as plastic stabilizers in PVC production Recoveries were observed in oyster popula- and as catalysts in some industrial processes. tions of Basin d’Arcachon (Alzieu, 1986) and in Environmental problems derived from the gastropods from coastal environments in Unit- use of organotin compounds as antifouling ed Kingdom (Evans et al., 1996; Tester et al., agents were first reported by the end of the 1996; Tester & Ellis, 1995). However, Kure & De- 1970s after a sharp decline in oyster produc- pledge (1994) reported that in Denmark, high tion was observed in the Basin d’Arcachon, concentrations could still be observed in filter France (Alzieu et al., 1986, 1989). The main bio- feeders years after establishment of the re- logical alterations initially ascribed to the wide- stricted use. spread use of organotins were balling in oysters Although TBT and TPT can be decomposed (Alzieu et al., 1986, 1989; Axiak et al., 1995; in the water column rather fast (half life time ~ Dyrinda, 1992), death of mollusk larvae (Hori- 7-15 days) (Foale, 1993), estimated half life of gushi et al., 1998) and imposition of male sexu- the order of 1 year for toxic sediments, to al characteristics to females of gastropods (El- decades for anoxic sediments, are indicative of lis & Pattisina, 1990; Fioroni et al., 1991; Gibbs relatively high persistence in these environ- & Bryan, 1987). This last effect, denominated ments, specially in the absence of oxygen (Dow- imposex is induced in several gastropod species son et al., 1996). In environments with high both in the field and in laboratory studies at particulate loads where photochemical reac- very low concentrations of TBT (imposex is ini- tions are less active and transport to sediments tiated at less than 1ng/L-1 TBT in Nucella la- may occur before extensive degradation in the pilus, and at 3-5ng/L-1 all females may be ster- water column takes place, sediments are a po- ilized (Gibbs & Bryan, 1994; concentrations of tential source of TBT and TPT even after the 1ng/L-1 can induce imposex development in use has been interrupted (Dowson et al., 1993, Thais clavigera, Horiguchi et al., 1995). This wide- 1996; Langston & Pope, 1995; Sarradin et al., ly observed effects suggests that TBT and TPT 1991, 1994). The tendency to accumulate in may also act as endocrinal disruptors (Horiguchi sediments becomes evident from the levels of et al., 1995, 1997a; Liu & Suen, 1996; Math- TBT reported for contaminated sediments that iessen & Gibbs, 1998; Oehlmann et al., 1996). may range from 300ng.g-1 up to 1,000ng.g-1 or The detrimental effects of TBT and TPT up- more, when compared to slightly contaminat- on non-target organisms led to restrictions to ed sediments, where levels are in the range of the use, first in France in 1982, where antifoul- 10 to 50ng.g-1 (Waite et al., 1991). Values as high ing paintings containing TBT were prohibited as 4,500ng.g-1 TBT in sediments where detect- for boats of less than 25m length (Sarradin et al., ed in Thailand (Kan-Atireklap et al., 1997a) or 1991; Waite et al., 1991), and then in many other 3,200ng.g-1 TBT in Hong-Kong (Ko et al., 1995). countries, such as the United Kingdom (1987), After 1994, reports from several authors on United States (1988), Sweden, New Zealand the presence of TBT in higher organisms in- (1989), , Japan (1990) and Denmark cluding cetaceans (Iwata et al., 1994; Kannan & (1991) (Champ & Pugh, 1987; de Mora et al., Falandysz, 1997; Tanabe, 1999), marine birds 1989; Horiguchi et al., 1994; Kure & Depledge, (Guruge et al., 1997a, 1997b) and humans (Kan- 1994; May et al., 1993; Stewart & de Mora, 1990; nan & Falandysz, 1997; Takahashi et al., 1999) Stuer-Lauridsen & Dahl, 1995). Germany and demonstrated that organotin compounds are Switzerland imposed restrictions also for fresh- likely to be transferred among different trophic water environments (Champ, 1986). Recently levels.

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Little is known about the behavior of organ- al., 1997) and invertebrates (mollusks and tuni- otin compounds in tropical marine environ- cates) (Cima et al., 1995; Morcillo & Porte, ments. Imposex has been observed in several 1997) call for epidemiological investigations species of gastropod on the coast of India (Cro- focused upon sensitive populations. nia konkanensis – Vishwa-Kiran & Anil, 1999); In Brazil, imposex in Thais haemastoma New Zealand (Lepsiella scobina, Thais orbita, was first reported by Magalhães et al. (1997), in Haustrum haustorium, Xymene ambiguus, Santos Bay, São Paulo, by Fernandez et al. Taron dubius, Cominella virgata, Amalda (Ba- (1998), in Guanabara Bay, Rio de Janeiro and by ryspira) australis – Stewart et al., 1992); Aus- Castro et al. (2000), in Fortaleza, Ceará. tralia (Lepsiella vinosa – Nias et al., 1993; T. or- The present work reports recent findings on bita – Foale, 1993); Thailand (Thais distin- possible TBT related effects in two of these ar- guenda, Thais bitubercularis, Morula musiva – eas of the Brazilian coast. Bech, 1999a, 1999b; Phalium bisulcatum, Dis- torsio reticularis, Murex occa, Murex trapa, Murex tribulus, Chicoreus capucinus, Thais gra- Methodology data, Thais lacera, Babylonia areolata, Nassar- ius livescens, Nassarius stolatus, Hemifusus ter- The development of imposex in T. haemastoma natanus, Voluma conchlidium, Cymbiola no- was used as a tool to search for indications of bilis, Melo melo – Swennen et al., 1997); Indone- biological stress derived from TBT and TPT in sia (Thais kieneri, Thais savignyi, tur- Guanabara Bay and in coastal areas of Fort- binellus – Evans et al., 1995); Malaysia (T. gra- aleza. The bioindicator was selected based on data, C. capucinus, T. clavigera, Thais jubilaea, the following properties: (1) ubiquity along the T. bitubercularis – Tan, 1997, 1999) and Hong Brazilian coast (Leal, 1984); (2) tendency to im- Kong (T. clavigera – Blackmore, 2000). In South posex development (Spence et al., 1990); (3) ex- America, imposex occurrence is reported in isting scientific evidences of imposex related to Chile (Chorus giganteus, Xanthochorus cassidi- TBT and TPT for organisms of the same formis, Nucella crassilabrum – Gooding et al., (T. clavigera and Thais bronni) (Horiguchi et 1999) and Brazil (Castro et al., 2000; Fernandez al., 1997a; Liu & Suen, 1996). et al., 1998; Magalhães et al., 1997). Information Figures 1 and 2 show the sampling stations on concentration in waters (Hashimoto et al., from which 20 to 50 specimens of T. haemas- 1998; Malaca straits), sediments (Kan-Atireklap toma were collected. Once in the laboratory, et al., 1997a – Thailand; Ko et al., 1995 – Hong the specimens were narcotized by immersion

Kong; Stewart & de Mora, 1992 – Fiji Island; in a 1:1 solution of local seawater and MgCl2 King et al., 1989 – New Zealand) and organisms 7%. Shell length was measured with the aid of a (bivalves, Perna viridis – Kan-Atireklap et al., vernier calliper and after removing the soft 1997b; Mytidus edulis and Aequipecten irradi- parts from the shell, sex was determined based ans – Guolan & Yong, 1995; gastropods, T. clav- on the presence (female) or absence (male) of igera, Thais tuberosa and Morula granulata – albumen and sperm ingestion glands (Figure 3) Liu et al., 1997) are scarce, although reported (Gibbs & Bryan, 1987; Horiguchi et al., 1994; levels specially in areas including intensive Spence et al., 1990). Penis length was also mea- navigation and installations have been sured in males and imposexed females as in high (over 1,000ng.g-1) and of the same order Axiak et al. (1995) e Stewart et al. (1992). as those available for temperate climate areas Imposex occurs at different levels which are (Morgan et al., 1998; Ruiz et al., 1998; Swennen related to TBT concentrations in water and in et al., 1997; ten Hallers-Tjabbes et al., 1994). As gastropod food; i.e., to the presence of this for the effect on humans, although organotin compound in the local environment. There- compounds are considered extremely toxic, fore, different levels of alterations may be dis- environmental routes of exposure are usually criminated, ranging from apparently unaffect- not considered very critical (Schweinfurth & ed females to those showing male attributes. In Günzel, 1987; Tsuda et al., 1995). However, the the present work, four indicators of imposex proved accumulation in filter feeders may be were applied to the determine different level of an important pathway to consumers (Guolan & imposex: (1)% of imposexed females; (2) rela- Yong, 1995). tive penis length index (RPLI); (3) relative penis Besides causing hormonal disruption, evi- size index (RPSI), and (4) vas deferens sequence dences that organotins seem to act upon the index (VDSI). RPLI and RPSI were calculated by 3 immune system of mammals (cetacean, rab- using the relations ([CFI/CM].100) and ([CFI / 3 bits, rats and sheep) (Dacasto et al., 1994a, CM ].100) (Gibbs & Bryan, 1987; Huet et al., 1994b; Kannan & Falandysz, 1997; Kannan et 1996; Minchin & Minchin, 1997), respectively,

Cad. Saúde Pública, Rio de Janeiro, 18(2):463-476, mar-abr, 2002 466 FERNANDEZ, M. A. et al.

Figure 1 criminating between different levels of alter- ations. VDSI is the only index that responds ef- Location of Guanabara Bay area and reference station in the coast of Rio de Janeiro fectively over a broad range of imposex. It is and monitoring stations along the coast of Fortaleza, Ceará State. useful in assessing the reproductive capacity of the studied population; populations with a mean VDSI above IV are comprised of sterile

35’ 38o30’ Atlantic Ocean females, and show reduced reprodutive capac- 1 2 ity (Gibbs & Brian, 1994; Oehlmann et al., 1996, Mucuripe Harbor 1998; Stroben et al., 1995). 8 3 4 7 VDSI was obtained through two different 5 Ceará River 6 approaches: (1) by using the scale of Gibbs (Gibbs & Bryan, 1987), and (2) by using a modi- fied scale of seven levels of imposex, developed

03o45’ by Fernandez et al. (1998). The development of 9 a modified scale was required because of the Fortaleza difficulty to discriminating among initial stages 10 • Coco River of the Gibbs scale without using microscopy and histological analysis. The new scale pro-

0 2 4 km vided an easy field method for assessing vas deferens sequence using only a magnifying Pacoti River glass. In this scale, level I of imposex was deter- mined as in Solé et al. (1998) while discrimina- tion between level III and IV was set according to Horiguchi et al. (1994). The other imposex levels were determined following the original scale of Gibbs. It should be emphasized that 44o00’ W 43o00’ W N this scale was originally developed for N. la- Guanabara Bay pilus. The same approach, however, could be adapted for other genera with little modifica- tions (Fioroni et al., 1991; Horiguchi et al., 1994; Solé et al., 1998; Stewart et al., 1992; Guaratiba Point Stroben et al., 1995). The modified scale shows

23o00’ S a slight tendency to underestimated results, Sepetiba Bay Cagarras Islands however, there is in general, general agreement Atlantic Ocean with the Gibbs scale (Table 1). The more de- tailed VDSI development scheme proposed by Fioroni et al. (1991) and Stroben et al. (1995), was tested, but histological analyses are re- quired for its application. •

Results and discussion

Imposex in Guanabara Bay, Rio de Janeiro

where CFI is the average penis length in impo- The results for the biomonitoring in Guana- sexed females and CM is the average penis bara Bay are shown in Table 2. Figures 2 and 4 length in males at each sampled station. show a comparison of the actual distribution The first index is a good indicator of impo- of T. haemastoma with that in the 1960s, and sex response at low environmental expositions, the geographic incidence of imposex, respec- when not all females show male attributes tively. The potential sources of TBT and TPT are (Curtis, 1994; Gibbs & Bryan, 1994). The penis depicted in Figure 3. development indexes, RPLI and RPSI, are more As can be observed from Figure 2, the actu- adequate for medium (RPLI) or high (RPSI) im- al distribution of T. haemastoma differs signifi- posex levels. cantly from that reported for the 1960s when In this last situation, penis length of impo- the use of TBT in several areas of the globe was sexed females approaches those of males, and initiated. The species nowadays is absent in all the RPLI index becomes less effective in dis- stations at the inner areas of the bay (stations

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1, 2, 3 and 4) even though the past occurrence Figure 2 of T. haemastoma in stations 2, 3 and 4 have been reported by Leal (1984). In the sampling Map showing the sampling stations and main sources of TBT and TPT of August 1999, only empty shells were found in Guanabara Bay. in station 1. 43 15’ 43 05’ Organisms were not found during the sam- 22 40’ pling campaigns of February and August 1999 in station 5, located under the central part of the Rio-Niterói Bridge, and in station 9 (1998) located in a sewage contaminated area near a marina. However, while in all these areas T. Guanabara Bay haemastoma was absent, the mussell Perna perna, a common prey for the gastropod, was abundant, unless for the bridge site where mussel is frequently harvested for human con- sumption. The same feature was observed in M stations 6 (presence of only one juvenile speci- CT men), 8 (presence of only two young organ- NB 22 50’ isms) and 12, which are located near a harbor SA and two marinas. The presence of young or- ganisms may indicate that a recolonization of the areas is possible, as reported by Spence et NB al. (1990) and Blackmore (2000) for species S NB having planktonic larvae. It should be noted that stations 9 and 12 are in sites of restricted 1960 water circulation, a condition that favors the M 1999 M build up of critical TBT levels in the water Leal, 1984 This study M (Gibbs & Bryan, 1994; Horiguchi et al., 1994, Stations 1997b; Nicholson et al., 1998). TBT/TPT sources Imposex indexes were generally high in the Itacoatiara 2Km >> stations located inside Guanabara Bay (Figure 23 00’ 4). The relative abundance of imposexed fe- << 60km Barra de Guaratiba males in a certain site is a tool for impact as- SA sessment when concentrations of TBT in water are relatively low and only a fraction of the fe- males is masculinized (Curtis, 1994; Gibbs & Bryan, 1994). For all sites inside Guanabara Bay M = marine; CT = commercial terminal; S = shipyard; NB = naval base; SA = ship anchoring area. Comparison between the actual occurrence of T. haemastoma and the 100% of the analyzed females showed some de- distribution observed in the 1960s in Guanabara Bay is also showed as shaded areas. gree of sexual alteration, and only in four sta- tions outside the bay, normal and less impo- sexed females were present (Ponta do Arpoa- dor, Itacoatiara, Cagarras Island and the con- Values over 100 indicate that imposexed fe- trol station at Barra de Guaratiba) (Figure 4). males mean penis lenghts (RPLI), or volumes RPLI and RPSI levels are shown in Figure 4. (RPSI) are greater than those of males from the Except for some stations (RPLI: 17b, 19 and 20; same locations. Nicholson et al. (1998) reported RPSI: 15, 17b, 18, 19 and 20) where due to the that values of RPSI in N. lapilus were in the small penis size in the females, indexes could range 37.9 to 44.8 near Sollun Voe, an oil tanker not be determined, all other stations inside port, and Spence et al. (1990) recorded values as Guanabara Bay showed measurable indexes. high as 48.3 to 81.7 in T. haemastoma collected RPSI and RPLI values above 30 and 70, respec- from and marinas in the Azores. tively, obtained for some inner sites of the bay An increasing gradient in imposex intensity have been considered high in other regions towards inner areas can be observed both in the known as polluted by TBT. For instance, values western (RPLI from N.A. to 106.3 and RPSI from ranging from 60.9 to 127.3 for RPLI and from N.A. to 107.6 in stations 11, 13, 15, 17, 20) and 22.2 to 206.2 for RPSI were recorded in T. clav- eastern (RPLI from N.A. to 107.6 and RPSI from igera and T. bronni (Horiguchi et al., 1994, N.A. to 125 in stations 7, 14, 16, 19) sides of the 1997b) from the most polluted sites in Japan, bay entrance. Those gradients include all impo- around Aburatsubo in the Miura Peninsula. sex levels so far detected in Guanabara Bay.

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Figure 3

Schematic representation of T. haemastoma. (A) male; (B) healthy female; (C) imposexed female.

A B C

os cg pt sig os me cg os me k sig sd tn me ag ag ts tn k tn k

pn v pn ov ov foot foot cm dg op op foot dg cm dg cm 1 3 cm op

tn – tentacles; me – mantle edge; os – osphradium; cm – collumela muscle; op – operculum; dg – digestive gland; k – kidney; pn – penis; ts – testicle; sd – spermatic duct; pr – prostate; v – vulva (inside mantle cavity); ov – ovary; ag – albumem gland; sig – sperm ingesting gland; cg – capsule gland.

Table 1

Comparison between the Gibbs scale and the adapted scale for vas deferens sequence index (VDSI).

Gibbs scale Characteristics of imposex development Modified scale

0 Normal Female 0 I Beginning of penis formation (A), Beginning of vas deferens formation (G) I II Formed penis, length < 2mm II III Formed penis, length > 2mm, distal vas deferens present (G) III (RPLIi < 0.5) IV Completely developed vas deferens IV (RPLIi > 0.5) V Vulva blocked by growth of vas deferens tissues V VI Dark mass of aborted capsules in capsule gland VI

RPLIi = Relative Penis Length Index of female i = [CFI i / CM] * 100 (adapted from Gibbs & Bryan, 1987).

The penis length is a better tool to charac- harvested during February 1999 while in Au- terize the first and medium stages of imposex gust 1999 only a few young organisms were development than the penis volume, therefore, available for sampling. In station 11, organisms RPLI values seems to be more adequate for dis- of VDSI V and VI were found in 1997 and in Jan- criminating between stations than RPSI, in uary 2000, only a single female amongst 18 Guanabara Bay. Similar findings were reported adults was present showing VDSI of IV. The un- by Horiguchi et al. (1994), in T.clavigera and T. balanced sex distribution in those stations may bronni along the Japanese coast. result from selective extinction of females due The observed VDSI values and existing im- to the blockage of capsule glands, a phenome- posex gradients (Figure 4) confirmed the ob- non observed in other regions and described servations given above. In stations 6 and 11 by Spence et al. (1990), Nicholson et al. (1998) sterile organisms with VDSI levels V and VI and Blackmore (2000). were found. In station 6 in Boa Viagem, Niterói, Based on the results obtained for the sever- only a female was found amongst 15 adults al indexes given above, we inferred that three

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Table 2

Results of biological monitoring in Guanabara Bay.

Station Date Location n (males/females) % imposex RPLI RPSI VDSI

1 August 1999 Paquetá Island (*) NF NA NA NA NA 2 March 1998 Valente (*) NF NA NA NA NA 3 March 1998 Zumbi Beach (*) NF NA NA NA NA 4 March 1998 Matoso Point (*) NF NA NA NA NA 5a February 1999 Rio-Niterói Bridge (*) NF NA NA NA NA 5b August 1999 Rio-Niterói Bridge (*) NF NA NA NA NA 6 February 1999 Feiticeiras Rocks 1 (NA) NA NA NA NA 7a February 1999 Boa Viagem Island (*) 15 (14/1) 100.0 107.6 125.0 V 7b August 1999 Boa Viagem Island (*) 10 (NA) NA NA NA NA 8 September 1997 Glória Marine (*) 2 (1/1) NA NA NA NA 9 March 1998 São Francisco Inlet (*) NF NA NA NA NA 10 September 1997 Flamengo Beach 8 (5/3) 100.0 106.3 120.0 IV 11a April 1997 Urca, São João Fortress (*) 27 (18/9) 100.0 90.3 79.0 IV 11b July 1997 Urca, São João Fortress (*) 29 (15/14) 100.0 90.8 75.0 V 11c January 2000 Urca, São João Fortress (*) 18 (17/1) 100.0 81.6 54.4 IV 11X January 2000 Urca, São João Fortress, internal area 21 (9/12) 100.0 21.8 1.1 I-III 12a April 1997 Rio de Janeiro Yatch Club NF NA NA NA NA 12b January 2000 Rio de Janeiro Yatch Club 1 (1/0) NA NA NA NA 13 May 1998 – March 2000 Vermelha Beach 188 (120/68) 100.0 44.0 13.0 III 14 July 1998 Adão Beach (*) 17 (12/5) 100.0 83.1 57.0 IV 15 July 1997 Leme Beach 17 (11/6) 100.0 8.5 < 0.1 I 16a July 1998 Itaipú Point (*) 15 (10/5) 100.0 32.7 1.4 II 16b July 1998 Itaipú Point (*) 17 (11/6) 100.0 37.6 13.0 II 17a May – August 1998 Arpoador Point (*) 26 (16/10) 30.0 44.1 8.6 II 17b January 2000 Arpoador Point (*) 20 (11/9) 22.2 < 0.1 < 0.01 I-II 18 May 1997 Barra de Guaratiba Beach 51 (26/25) 19.0 5.6 <0.1 I 19 March 2000 Itacoatiara Beach (*) 30 (15/15) 53.3 < 0.1 < 0.01 0-I 20 April 2000 Cagarras Islands 27 (12/15) 40.0 < 0.1 < 0.01 0-I

NF = not found; NA = Not analyzed; (*) = Animal reported before in this station; RPLI = Relative penis length index; RPSI = Relative penis size index; VDSI = Vas deferens sequence index.

regions, with different degrees of impact exist ability that can be ascribed to the difference in in Guanabara Bay: (1) the region extending size and sexual development of the organisms from the Rio-Niterói Bridge to the northern available for sampling. This was the greatest area of the bay (stations 1 to 4) where presently variability observed in repeated sampling of the bioindicator is not available. Here, at the the same station. present stage of knowledge, it is difficult to at- The water circulation pattern and the prox- tribute the absence of organisms to TBT or TPT imity to possible sources of TBT seem to be im- contamination because this region receives portant factors controlling the degree of impo- heavy loads of other pollutants derived from sex in the studied area. This trend to higher im- domestic and industrial sources; (2) the region posex indexes (RPSI > 10; RPLI >30; VDSI ≥ II) between the Rio-Niterói Bridge and the mouth with increasing boat traffic or anchoring and of the bay (stations 5 to 14) where high indexes water stagnation is a typical feature of TBT of imposex and strong imposex gradients were contamination (Axiak et al., 1995; Smith & found, and (3), regions outside of Guanabara McVeagh, 1991; Spence et al., 1990; Stewart et Bay (stations 15 to 20) in which only organisms al., 1992) that has been used to map zones of with low imposex levels were found, and lesser influence in a regional (Minchin et al., 1996; impact is to be expected. Minchin & Minchin, 1997) and national scale The results of the two samplings in Arpoa- (Horiguchi et al., 1994, 1997b; ten Hallers- dor (station 17) showed a wide range of vari- Tjabbes et al., 1994, 1996). A few authors

Cad. Saúde Pública, Rio de Janeiro, 18(2):463-476, mar-abr, 2002 470 FERNANDEZ, M. A. et al.

Figure 4a

Intensity of imposex in Guanabara Bay as determined according to RPLI index. Displayed area corresponds to the actual area where T. haemastoma is still found in the bay.

N.E.

107.6 São Francisco Inlet

N.E. Glória Marine N.E. 106.3 90.8

Flamengo Beach Urca

Adão Beach Rio de Janeiro Yatch Club N.E. Vermelha Beach 83.1 44.0 8.5 37.6 Leme Beach Itaipu Point N.A. 2km

N.A. – 44.1

60km N.A. 5.6

Figure 4b

Intensity of imposex in Guanabara Bay as determined according to RPSI index. Displayed area corresponds to the actual area where T. haemastoma is still found in the bay.

N.E.

125 São Francisco Inlet

N.E. Glória Marine N.E. 120 79

Flamengo Beach Urca

Adão Beach Rio de Janeiro Yatch Club N.E. Vermelha Beach 57 13 < 0.1 13 Leme Beach Itaipu Point N.A. 2km

N.A. – 8.6

60km N.A. < 0.1

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Figure 4c

Intensity of imposex in Guanabara Bay as determined according to VDSI index. Displayed area corresponds to the actual area where T. haemastoma is still found in the bay.

N.E.

125 São Francisco Inlet

N.E. Glória Marine N.E. IV IV

Flamengo Beach Urca

Adão Beach Rio de Janeiro Yatch Club N.E. Vermelha Beach IV III I II Leme Beach Itaipu Point 0 – I 2km

0 – II

60km 0 – I I

(Davies et al., 1987; Evans, 1999; Nicholson et of imposex in stage I (VDSI) was observed in al., 1998) based on experimental observations females that showed a small protuberance as a have proposed that TBT impact would be re- first step towards penis development. Because stricted to sites directly under influence of spe- in this initial stage, the penis dimensions can- cific sources showing, therefore, little tendency not be measured, RPSI was not determined for to migrate over long distances from the input this station, neither was it for Formosa and area. Our results seems to confirm this obser- Poço da Draga, where females displayed simi- vation. In Guanabara Bay the actual absence of lar alterations. In station 3 (Barra do Ceará), the organism in previously reported sites is an advanced stages of imposex were found with additional evidence of impact (Huet et al., VDSI ranging between II and III, a RPLI of 31.9 1996; Minchin & Minchin, 1997; Smith, 1996) and a RPSI of 3.2. In this area there is a small that may be derived from TBT, especially when shipyard and boat cruising is intensive. In Mu- mussels are abundant (Morcillo & Porte, 1997). curipe Beach (station 6), near Mucuripe Har- bor and Mansa Beach (station 7), also close to Imposex in the coast area the harbor, where boat traffic associated to of Fortaleza, Ceará fishing and tourism as well as large ship traffic are very intense, VDSI was V, except for a single Imposex was registered in several of the sam- female in Mansa Beach that had VDSI equal to pling sites (Figure 1 and Table 3) and the higher IV. The values of the RPLI and RPSI obtained indexes were found in females collected from for both station 6 (RPLI 63.6 and RPSI 25.7) and areas near the Mucuripe Harbor. In the Pache- station 7 (RPLI 82.8 and RPSI 61.7) are compa- co Beach and in Titanzinho, Caça e Pesca and rable to those found in Guanabara Bay and Sabiaguaba, beaches where boat cruises are elsewhere, and support the conclusions drawn virtually absent, no imposex or related anom- from the vas deferens index. alies were observed. The absence of imposex in Contrary to the observations of Bryan et al. the last three beaches located east of Mucuripe (1986) in N. lapillus whose populations had se- Harbor is due to the water circulation in the re- verely decreased prior to the establishment of gion that prevents contamination predomi- restrictions to TBT use, populations of T. hea- nantly flowing from east to west (Maia, 1998). mastoma in Fortaleza were apparently stable In station 2 (Dois Coqueiros Beach) evidence and abundant in all sampling sites. The intra-

Cad. Saúde Pública, Rio de Janeiro, 18(2):463-476, mar-abr, 2002 472 FERNANDEZ, M. A. et al.

Table 3

Results of biological monitoring in Fortaleza, Ceará.

Station Date Location n (males/females) % imposex RPLI RPSI VDSI

1 March 2000 Pacheco Beach 27/23 0.0 0.0 0 0 2 March 2000 Dois Coqueiros Beach 20/30 100.0 < 0.1 < 0.01 I-II 3 October 2000 Barra do Ceará Beach 15/35 100.0 31.9 3.2 III 4 November 1999 Formosa Beach 26/24 100.0 < 0.1 < 0.01 I 5 December 1999 Poço da Draga Beach 32/18 100.0 < 0.1 < 0.01 I 6 September 1999 Mucuripe Beach 36/14 100.0 63.6 25.7 IV 7 March 2000 Mansa Beach 32/18 100.0 82.8 61.7 V 8 January 2000 Titanzinho Beach 25/25 0.0 0.0 0.0 0 9 January 2000 Caça e Pesca Beach 22/28 0.0 0.0 0.0 0 10 December 1999 Sariguaba Beach 20/30 0.0 0.0 0.0 0

RPLI = Relative penis length index; RPSI = Relative penis size index; VDSI = Vas deferens sequence index.

capsular development of N. lapillus (Bauchet, served sites, the distribution of imposex levels 1989; Feare, 1970) seems to favor population points towards a possible cause-effect relation decline in polluted sites because organisms are with TBT and/or TPT. directly exposed to the contaminant since the The biomonitoring results for Guanabara early stages of life. As T. haemastoma develop- Bay and Fortaleza show that higher imposex ment presents a stage of planktonic larvae, ar- indexes are occurring in areas of harbor and eas can be re-colonized by organisms import- marina activities, confirming the trend ob- ed from uncontaminated sites (Spence et al., served by other authors in several regions of 1990) promoting constant provision of healthy the globe. Because Guanabara Bay is subjected individuals. to various sources of organic and inorganic T. haemastoma spawning was observed pollutants, it is difficult to establish unambigu- even in sites where stage IV of VDSI occurred, ous cause-effect relationships. However, the both in Fortaleza and in Guanabara Bay. A sim- actual absence of T. Haemastoma in areas ilar observation was reported by Gibbs & Bryan where it was commonly found in the past, rais- (1986), Fioroni et al. (1991) and Oehlmann et es concern on the extension of possible effects al. (1996) that concluded reproductive failure of TBT contamination in the bay. will only occur in organisms displaying VDSI All sampled females from Guanabara Bay above V, a limit above which blockage of the fe- displayed some level of imposex. Differently, in male genitals initiates. In Mansa Beach, how- Fortaleza some sites were unaffected and, pos- ever, the majority of the sampled organisms sibly due to the lower level of contamination of showed VDSI V with penis and vas deferens the marine environment, no evidence of ex- completely formed and blocked genital cavity. tinction were registered. In this case spawning was not observed, sug- The wide distribution of T. haemastoma gesting that affected females were unable to re- along the Brazilian coast makes the species a produce. good candidate for large scale biological screening of “hot spots” of TBT and TPT pollu- tion, although further laboratory and field Conclusions studies are required to establish a quantitative relation between imposex level and concentra- In the present investigation, sexual alterations tion of TBT and/or TPT in the marine environ- leading to imposex in females of neogastropod ment. T. Haemastoma were observed in Rio de Janeiro and in Ceará coastal areas. This reprodutive anomaly has been attributed to the presence of TBT and TPT in the water (Gibbs & Bryan, 1994; Horiguchi et al., 1997a, 1997b; ten Hallers- Tjabbes et al., 1994). Although concentration levels in water are not yet available for the ob-

Cad. Saúde Pública, Rio de Janeiro, 18(2):463-476, mar-abr, 2002 POSSIBLE EVIDENCE OF ENVIRONMENTAL CONTAMINATION BY ORGANOTIN COMPOUNDS 473

Acknowledgements

The authors are grateful to Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro for financial sup- port and to L. Gebara, G. Telles, L. Bispo, P. Miyamo- to, P. Nogueira, L. Varoveska, G. Uller, C. A. Meirelles and to Projeto Maqua/Universidade do Estado do Rio de Janeiro for the collaboration in the field work. L. P. Maia, from Departamento de Geologia, Universidade Federal do Ceará, kindly furnished information on coastal currents in Fortaleza. We also thank J. Oehl- mann, M. Bech and M. Solé for very useful discus- sions about the development of this work.

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Cad. Saúde Pública, Rio de Janeiro, 18(2):463-476, mar-abr, 2002